Age-induced strength loss, dynapenia, is accentuated in females due to estradiol (E2) deficiency that naturally occurs with aging. The overall goal of this project remains on determining the cellular and molecular mechanisms through which E2 deficiency perturbs muscle and myosin contractile functions and how E2 improves strength in aging females. This is a competitive renewal submission of a funded proposal that continues to produce exciting discoveries and numerous publications. Results from the previous funding periods have led to the novel hypotheses outlined in this proposal. Aim 1 tests the hypothesis that E2 deficiency causes loss of muscle strength due to compromised phosphorylation of contractile proteins impairing force generation as well as affecting the myosin super relaxed state during relaxation. Furthermore, it is predicted that treatment with physiological levels of E2 rescues strength through activation of the ? estrogen receptor (ER?) and the G protein ER (GPER) and downstream activation of key kinases. Innovative in vivo experimental approaches and contemporary phosphoproteomics techniques, combined with manipulation of E2 and ERs pharmacologically, surgically, and genetically will be used to deduce estrogenic mechanisms acting on aging muscle. Skeletal muscle endures repetitive injury throughout life and aging as well as E2 deficiency impair the recovery of strength following such injury. In Aim 2, a systematic review and meta-analysis of the literature paired with experimental testing of E2 treatment in ovariectomized adult and ovarian-senescent, aged mice will address the hypothesis that muscle inflammation, necessary for recovery of strength from injury, is enhanced with physiological levels of E2 but blunted with supraphysiological levels. Aim 2 also tests the hypothesis that E2 deficiency disrupts neutrophil functions in injured muscle, and will utilize state-of-the-art mass cytometry (CyTOF) to determine E2 responsiveness of other inflammatory cells in injured muscle, identifying those cells that release E2-sensitive chemokines/cytokines as well. Completion of these aims will culminate in substantial contributions to our knowledge of aging skeletal muscle, especially in females. Specifically, results will provide clinically-relevant information about estrogenic treatments beyond those for reproductive tissues with the goal of understanding how E2 can most effectively maintain muscle strength and movement quality in aging women.